The subject of the present invention is complexes formed by the inclusion of precursors, from a natural source, of organic isothiocyanates in cyclodextrins.
It particularly concerns the solubilisation and/or controlled release of bacteriostatic, bactericidal and/or fungicidal agents formed of organic isothiocyanates, from such complexes.
It also concerns bacteriostatic, bactericidal and/or fungicidal compositions containing such complexes, which can be used in the fields of food and food processing.
Organic isothiocyantes are known to have remarkable bacterio- and fungistatic properties, and their activity in controlling the proliferation of aerobic and anaerobic micro-organisms has been well identified, as described in xe2x80x9cAntimicrobial Properties of Isothiocyanates in Food Preservationxe2x80x9d by P. J. Delaquis and G. Mazza, published by James Giese in xe2x80x9cFood technologyxe2x80x9d, November 1995, pages 73 to 84 [1].
However, their physical properties (they are most often volatile liquids) make their use very difficult in food and food processing applications since it is impossible to control the minimal doses which ensure the preservation process without harming the organoleptic properties of the food to be preserved.
The precise subject of this present invention is bacteriostatic, bactericidal and/or fungicidal compositions in which the organic isothiocyanate is stabilised in the form of an inclusion complex in a cyclodextrin, permitting its solubilisation in an aqueous medium and control over its release.
According to the invention, the inclusion complex is formed of a natural precursor of an organic isothiocyanate included in a cyclodextrin.
According to the invention, the organic isothiocyanate preferably meets the formula:
R1xe2x80x94Nxe2x95x90Cxe2x95x90Sxe2x80x83xe2x80x83(I)
in which R1 represents an alkyl group, whether linear or branched, with 1 to 6 carbon atoms; an alkenyl group, linear or branched, with 2 to 6 carbon atoms; an arylalkyl group in which the alkyl group has 1 to 6 carbon atoms; a group having the formula: 
in which q=1 or 2 and R2 represents a hydrogen atom or an alkyl or alkoxyl group with 1 to 3 carbon atoms, the substituent R2 being in ortho, para or meta position; a group with the formula R3CO(CH2)nxe2x80x94 in which R3 is an alkyl group with 1 to 3 carbon atoms and n is a whole number of 3 or 4; a group with the formula R3OCO(CH2)nxe2x80x94 in which R3 and n are as defined above; a methylthioalkyl group with the formula CH3S(CH2)pxe2x80x94 in which p is a whole number from 1 to 10; or a group with the formula CH3xe2x80x94Sxe2x80x94CHxe2x95x90CHxe2x80x94(CH2)rxe2x80x94 in which r is a whole number from 1 to 8.
As an example of such isothiocyanates, mention may be made of those for which R1 has the denotation given in the appended table.
The natural precursor of organic isothiocyanate, or glucosinolate, may meet the formula: 
in which R1 has the denotation given above.
These natural precursors may in particular be those mentioned in the appended table.
These natural precursors may be obtained from plant sources such as those also listed in the appended table.
The cyclodextrin used in the inclusion complex described above may be a natural cyclodextrin or a chemically modified cyclodextrin.
It is recalled that the cyclodextrins or cyclomaltooligosaccharides are compounds of natural origin, formed by the chain conformation of glucose units bonded at xcex1-1.4.
The natural cyclodextrins are xcex1-cyclodextrin which contains 6 glucose units, xcex2-cyclodextrin which contains 7 glucose units and xcex3-cyclodextrin which contains 8 glucose units. Much research work has shown that these cyclodextrins could form inclusion complexes with hydrophobic molecules, thereby permitting their solubilisation in aqueous media and their use in various industries, as described in the work entitled: xe2x80x9cCyclodextrins and their Industrial Usesxe2x80x9d by D. Duchxc3xaane published by Editions de Santxc3xa9, 1987, pages 299 to 326, [2].
According to the invention, not only a natural cyclodextrin may be used, but also a natural cyclodextrin that is chemically modified through the addition of appropriate substituents. Said modified cyclodextrin may meet the formula: 
in which m=6, 7 or 8 and the R4s which may be identical or different, represent OH or a group chosen from among the X-alkylated or X-arylated groups in which X represents a carbon atom or a heteroatom provided that at least one of the R4s is not OH.
In this case, the substituents R4 of cyclodextrin are chosen such as to improve its properties, in particular in respect of the solubilisation and stabilisation of the organic isothiocyanates.
According to the invention, the inclusion complex of a natural precursor of an organic isothiocycate in a cyclodextrin, using an aqueous solution of cyclodextrin, may be prepared by the addition of the natural precursor to this solution. The cyclodextrins used are those previously described.
In this manner, the inclusion complex may be formed either in the form of an aqueous solution, or in the form of a precipitate which is subsequently separated from the solution. In this latter case, an aqueous solution saturated with cyclodextrin may be used.
According to the invention, the inclusion complexes of natural precursors of organic isothiocyanates in cyclodextrins may be used to prepare an inclusion complex of an organic isothyanate in a cyclodextrin.
Indeed, it is known that these natural precursors of organic isothiocyanates may be converted into the corresponding isothiocyanates by hydrolytic reaction with myrosinase in accordance with the following reaction diagram: 
Myrosinase is an enzyme naturally present in Cruciferae. The enzymatic hydrolysis of the natural precursor of formula (IV) by myrosinase leads to the corresponding organic isothiocyanate and to the release of a molecule of D-glucose and potassium acid sulphate.
According to the invention, it has been discovered that it is possible to prepare an inclusion complex containing organic isothiocyanate from an inclusion complex containing the natural precursor of this isothiocyanate through the action of myrosinase on this complex.
Therefore, a further object of this invention is a method for preparing an inclusion complex formed of an organic isothiocyanate included in a cyclodextrin, which comprises the following steps:
a) preparing, in an aqueous solution, an inclusion complex containing a natural precursor of an organic isothiocyanate in cyclodextrin, and
b) submitting the complex so obtained to the action of myrosinase in aqueous solution to convert the natural precursor included in the cyclodextrin into the corresponding organic isothiocyanate.
In this case, the aqueous solution used in the second step, and optionally in the first step, is a solution suitable for hydrolysis by myrosinase. It may be a buffer solution such as a phosphate buffer solution with a pH value of 6.
A further object of the invention is a bacteriostatic, bactericidal and/or fungicidal composition, made up of a dry mixture containing:
a) an inclusion complex of a natural precursor of an organic isothiocyanate, having bactericidal, bacteriostatic and/or fungicidial properties, in a cyclodextrin, and
b) myrosinase.
By way of example, the composition may be in powder, granule, wettable powder form etc. This composition may comprise appropriate vehicles and additives such as those generally used for the production of bacteriostatic, bactericidal and/or fungicidal products intended for the food or food processing industries.
It may be prepared using conventional methods.
With this composition, at the time of use, when the dry mixture is placed in contact with water or a humid atmosphere, the presence of water will convert the natural precursor present in the inclusion complex into the corresponding organic isothiocyanate through enzymatic hydrolysis with the myrosinase in the composition.
In this case, the composition is for example in powder form, or wettable powder form.